Apparatus, system, and method for collecting a target material

ABSTRACT

This disclosure is directed to an apparatus, system and method for retrieving target material from a suspension. A system includes a processing vessel, a displacement fluid, and a tube. The tube includes a funnel, a cannula, and a cavity. The cannula allows for fluid communication between the funnel and the cavity, such that the processing vessel is inserted into the cavity, and the cannula extends into the processing vessel.

CROSS-REFERENCE TO A RELATED APPLICATION

This application claims the benefit of Provisional Application No.62/345,172, filed Jun. 3, 2016, and is also a continuation-in-part ofapplication Ser. No. 14/610,522, filed Jan. 30, 2015, which claims thebenefit of Provisional Application No. 61/935,457, filed Feb. 4, 2014,and is also a continuation-in-part of application Ser. No. 14/495,449,filed Sep. 24, 2014 (now U.S. Pat. No. 9,039,999, issued May 26, 2016),which is a continuation-in-part of application Ser. No. 14/090,337,filed Nov. 26, 2013, which claims the benefit of Provisional ApplicationNo. 61/732,029, filed Nov. 30, 2012; Provisional Application No.61/745,094, filed Dec. 21, 2012; Provisional Application No. 61/791,883,filed Mar. 15, 2013; Provisional Application No. 61/818,301, filed May1, 2013; and Provisional Application No. 61/869,866, filed Aug. 26,2013; and is also a continuation-in-part of application Ser. No.14/266,939, filed May 1, 2014, which claims the benefit of ProvisionalApplication No. Provisional Application No. 61/818,301, filed May 1,2013, Provisional Application No. 61/869,866, filed Aug. 26, 2013, andProvisional Application No. 61/935,457, filed Feb. 4, 2014.

TECHNICAL FIELD

This disclosure relates generally to density-based fluid separation and,in particular, to retrieving target material from a suspension.

BACKGROUND

Suspensions often include materials of interests that are difficult todetect, extract and isolate for analysis. For instance, whole blood is asuspension of materials in a fluid. The materials include billions ofred and white blood cells and platelets in a proteinaceous fluid calledplasma. Whole blood is routinely examined for the presence of abnormalorganisms or cells, such as ova, fetal cells, endothelial cells,parasites, bacteria, and inflammatory cells, and viruses, including HIV,cytomegalovirus, hepatitis C virus, and Epstein-Barr virus. Currently,practitioners, researchers, and those working with blood samples try toseparate, isolate, and extract certain components of a peripheral bloodsample for examination. Typical techniques used to analyze a bloodsample include the steps of smearing a film of blood on a slide andstaining the film in a way that enables certain components to beexamined by bright field or fluorescence microscopy.

On the other hand, materials of interest that occur in a suspension withvery low concentrations are especially difficult if not impossible todetect and analyze using many existing techniques. As a result,practitioners, researchers, and those working with suspensions continueto seek systems and methods for accurate analysis of suspensions for thepresence or absence rare materials of interest.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B show an example tube.

FIGS. 2A-2C show an example tube-processing vessel system.

FIGS. 3A-3B show an example tube.

FIGS. 4A-4B show an example tube.

FIGS. 5A-5G show example sealing rings.

FIG. 6A shows a flow diagram of an example method for retrieving targetmaterial.

FIG. 6B shows a flow diagram of an example method for retrieving targetmaterial.

FIGS. 7A-7F show an example system retrieving target material.

FIGS. 8A-8E show an example system retrieving target material.

DETAILED DESCRIPTION

This disclosure is directed to an apparatus, system and method forretrieving target material from a suspension. A system includes aprocessing vessel, a displacement fluid, and a tube. The tube includes afunnel, a cannula, and a cavity. The cannula allows for fluidcommunication between the funnel and the cavity, such that theprocessing vessel is inserted into the cavity, and the cannula extendsinto the processing vessel.

FIG. 1A shows an isometric view of a tube 100. FIG. 1B shows across-sectional view of the tube 100 taken along the line I-I shown inFIG. 1A. The tube 100 may be any appropriate size or cross-sectionalshape (e.g. cylindrical, elliptical, triangular, square, rectangular, orthe like. The tube 100 includes a main body 102 having a sidewall whichconnects a first end 104, a second end 118, and a collection segment 106between the first end 104 and the second end 118. The tube 100 may alsoinclude a cap 108 to seal or close the first end 104, which may be open.The collection segment 106 includes a funnel 110, a cannula 112, and acavity 114.

The funnel 110 tapers away from the first end 104 towards the second end118. The funnel 110 channels target material from a mouth of the funnel110 into the cannula 112 which is connected to, and in fluidcommunication with, an apex of the funnel 110. The apex of the funnel110 has a smaller diameter than the mouth of the funnel 110. The funnel110 is formed by a tapered wall that may be straight, curvilinear,arcuate, or the like. The funnel 110 may be any appropriate shape,including, but not limited to, tubular, hemispherical, parabolic,conical, rectangular, pyramidal, or the like.

The cannula 112, such as a tube or a needle, including, but not limitedto a non-coring needle, extends from the apex of the concave opening 114and into the cavity 114. The cavity 114 is a concave opening extendingfrom an aperture 120 in the second end 118 towards the first end 104.The cavity 114 may accept and support a processing vessel (not shown).The cavity 114 may be any appropriate depth to accept and support theprocessing vessel (not shown). The cavity 114 may be threaded to engagea threaded portion of the processing vessel (not shown). The cannula 112may extend any appropriate distance into the cavity 114 in order toextend into, to puncture the base of, or be inserted into, theprocessing vessel (not shown). The cannula 112 may include a flat tip, abeveled tip, a sharpened tip, or a tapered tip. Furthermore, the cavity114 may be any appropriate shape, including, but not limited to,tubular, hemispherical, parabolic, conical, rectangular, pyramidal, orthe like. The cannula 112 can be composed of a variety of differentmaterials including, but not limited to, a ceramic; metals; organic orinorganic materials; and plastic materials, such as a polypropylene,acrylic, polycarbonate, or the like; and combinations thereof. Thecannula 112 may have a tip along a longitudinal axis of the cannula.

The first end 104 is sized to receive a cap 108. The cap 108 may becomposed of re-sealable rubber or other suitable re-sealable materialthat can be repeatedly punctured with a needle or other sharp implementto access the contents stored in the tube 100 interior and re-seals whenthe needle or implement is removed. Alternatively, the cap 108 may be ascrew cap with threads to engage complementary threads within or aroundthe first end 104. Alternatively, a clip may be placed over the cap 108and the first end 104 of the tube 100 to increase the pressure exertedby the cap 108 on the first end 104 to provide a more secure fit. Theclip may be a two-piece ring, a one piece ring wrapping around the fullcircumference of the tube 100, or a one piece ring wrapping around lessthan the full circumference of the tube 100, such as one-half (½),five-eighths (⅝), two-thirds (⅔), three-quarters (¾), seven-eighths (⅞),or the like. Alternatively, the cap 108 may be temporarily orpermanently affixed to the main body 102 of the tube 100 via the firstend 104, such as by welding (e.g. ultrasonic welding), adherence (e.g.an adhesive), clamping (e.g. a sealing ring, crimper, clamp, or thelike), or any other appropriate manner for temporarily or permanentlyaffixing two pieces.

The tube 100 may also include a plug 116 to be inserted into the cavity114 to inhibit fluid from escaping from the cannula 112 when the cannula112 is pointed downward. The plug 116 may be composed of re-sealablerubber or other suitable re-sealable material that can be repeatedlypunctured with the cannula 112 and re-seals when the cannula 112 isremoved. Alternatively, the plug 116 may be a screw cap with threads toengage complementary threads within the cavity 114 or outside or on thesecond end 118. Alternatively, a clip may be placed over the plug 116and the first end 104 of the tube 100 to increase the pressure exertedby the plug 116 on the first end 104 to provide a more secure fit. Theclip may be a two-piece ring, a one piece ring wrapping around the fullcircumference of the tube 100, or a one piece ring wrapping around lessthan the full circumference of the tube 100, such as one-half (½),five-eighths (⅝), two-thirds (⅔), three-quarters (¾), seven-eighths (⅞),or the like.

The tube 100 can be composed of a variety of different materialsincluding, but not limited to, a ceramic; metals; organic or inorganicmaterials; and plastic materials, such as polyoxymethylene (“Delrin®”),polystyrene, acrylonitrile butadiene styrene (“ABS”) copolymers,aromatic polycarbonates, aromatic polyesters, carboxymethylcellulose,ethyl cellulose, ethylene vinyl acetate copolymers, nylon, polyacetals,polyacetates, polyacrylonitrile and other nitrile resins,polyacrylonitrile-vinyl chloride copolymer, polyamides, aromaticpolyamides (“aramids”), polyamide-imide, polyarylates, polyaryleneoxides, polyarylene sulfides, polyarylsulfones, polybenzimidazole,polybutylene terephthalate, polycarbonates, polyester, polyester imides,polyether sulfones, polyetherimides, polyetherketones,polyetheretherketones, polyethylene terephthalate, polyimides,polymethacrylate, polyolefins (e.g., polyethylene, polypropylene),polyallomers, polyoxadiazole, polyparaxylene, polyphenylene oxides(PPO), modified PPOs, polystyrene, polysulfone, fluorine containingpolymer such as polytetrafluoroethylene, polyurethane, polyvinylacetate, polyvinyl alcohol, polyvinyl halides such as polyvinylchloride, polyvinyl chloride-vinyl acetate copolymer, polyvinylpyrrolidone, polyvinylidene chloride, specialty polymers, polystyrene,polycarbonate, polypropylene, acrylonitrite butadiene-styrene copolymer,butyl rubber, ethylene propylene diene monomer, and combinationsthereof. The tube 100 may be rigid or flexible.

FIG. 2A shows an exploded view of the example tube 100 and processingvessel 202. FIG. 2B shows an isometric view of the processing vessel 202inserted into the cavity 114 at the collection segment 106 of the tube100 to form a tube-processing vessel system 200. FIG. 2C shows across-sectional view of the processing vessel 202 inserted into thecavity 114 at the collection segment 106 of the tube 100 taken along theline II-II shown in FIG. 2B. The tube 100 and processing vessel 202 forma tube-processing vessel system 200. The processing vessel 202 may be aneppendorf tube, a syringe, or a test tube, and has a closed end 204 andan open end 206. The open end 206 is sized to receive a cap 208. The cap208 may be composed of re-sealable rubber or other suitable re-sealablematerial that can be repeatedly punctured with a needle or other sharpimplement to access the contents stored in the processing vessel 202interior and re-seals when the needle or implement is removed.Alternatively, the processing vessel 202 may also have two open endsthat are sized to receive caps. The processing vessel 202 may have atapered geometry that widens or narrows toward the open end 206; theprocessing vessel 202 may have a generally cylindrical geometry; or, theprocessing vessel 202 may have a generally cylindrical geometry in afirst segment and a cone-shaped geometry in a second segment, where thefirst and second segments are connected and continuous with each other.Although at least one segment of the processing vessel 202 has acircular cross-section, in other embodiments, the at least one segmentcan have elliptical, square, triangular, rectangular, octagonal, or anyother suitable cross-sectional shape. The processing vessel 202 can becomposed of a transparent, semitransparent, opaque, or translucentmaterial, such as plastic or another suitable material. The processingvessel includes a central axis 214, which when inserted into the cavity114 is coaxial with a central axis of the tube 100. The processingvessel 202 may also include a plug 210 at the closed end 204 to permitthe introduction of the target material or to exchange the targetmaterial with a displacement fluid 212. The closed end 204 may bethreaded to provide for a threaded connection with a threaded cavity(not shown) of the tube 100. The processing vessel 202 may be composedof glass, plastic, or other suitable material.

The plug 210 may be composed of re-sealable rubber or other suitablere-sealable material that can be repeatedly punctured with a needle orother sharp implement to access the contents of the processing vessel202 interior or permit introduction of contents into the processingvessel 202 and re-seals when the needle or implement is removed. Theplug 210 may be inserted into the processing vessel 202 such that a sealis maintained between the plug 210 and the processing vessel 202, suchas by an interference fit. Alternatively, the plug 210 can be formed inthe closed end 204 of the processing vessel 202 using heated liquidrubber that can be shaped while warm or hot and hardens as the rubbercools. An adhesive may be used to attach a plug 210 to the inner wall ofthe processing vessel can be a polymer-based adhesive, an epoxy, acontact adhesive or any other suitable material for bonding or creatinga thermal bond. Alternatively, the plug 210 may be injected into theprocessing vessel 202. Alternatively, the plug 210 may be thermallybonded to the processing vessel 202.

The cannula 112, for example, may have a tapered tip that punctures theplug 210 and extends into an inner cavity of the processing vessel 202with the shaft of the cannula 112 not extending into the inner cavity ofthe processing vessel 202. As explained in greater detail below, theinner cavity of the processing vessel 202 holds the target material. Thecannula 112 may be covered by a resealable sleeve (not shown) to preventthe target material from flowing out unless the processing vessel 202 isin the cavity 114 to a depth that allows the cannula 112 to justpenetrate the processing vessel 202. The resealable sleeve (not shown)covers the cannula 112, is spring-resilient, can be penetrated by thecannula 112, and is made of an elastomeric material capable ofwithstanding repeated punctures while still maintaining a seal.

The processing vessel 202 may be loaded with a displacement fluid 212prior to insertion into the tube 100. The displacement fluid 212displaces the target material, such that when the processing vessel 202is inserted into the primary vessel tube 100, which includes the targetmaterial, and the tube 100 and the processing vessel 202 undergocentrifugation, the displacement fluid 212 flows out of the processingvessel 202 and into the tube 100, and, through displacement, such asthrough buoyant displacement (i.e. lifting a material upwards), pushesthe target material through the cannula 112 and into the processingvessel 202.

The displacement fluid 212 has a greater density than the density of thedesired target material of the suspension (the density may be greaterthan the density of a subset of suspension fractions or all of thesuspension fractions) and is inert with respect to the suspensionmaterials. For example, the displacement fluid may have a density thatis approximately 0.001 to approximately 0.005 g/cm³ greater than thedensity of the desired target material. The displacement fluid 212 maybe miscible or immiscible in the suspension fluid. Examples of suitabledisplacement fluids include, but are not limited to, solution ofcolloidal silica particles coated with polyvinylpyrrolidone (e.g.Percoll), polysaccharide solution (e.g. Ficoll), iodixanol (e.g.OptiPrep), an organic solvent, a liquid wax, an oil, a gas, andcombinations thereof; olive oil, mineral oil, silicone oil, immersionoil, mineral oil, paraffin oil, silicon oil, fluorosilicone,perfluorodecalin, perfluoroperhydrophenanthrene, perfluorooctylbromide,and combinations thereof; organic solvents such as 1,4-Dioxane,acetonitrile, ethyl acetate, tert-butanol, cyclohexanone, methylenechloride, tert-Amyl alcohol, tert-Butyl methyl ether, butyl acetate,hexanol, nitrobenzene, toluene, octanol, octane, propylene carbonate,tetramethylene sulfones, and ionic liquids; polymer-based solutions;surfactants; perfluoroketones, such as perfluorocyclopentanone andperfluorocyclohexanone, fluorinated ketones, hydrofluoroethers,hydrofluorocarbons, perfluorocarbons, perfluoropolyethers, silicon andsilicon-based liquids, such as phenylmethyl siloxane; and combinationsthereof.

The processing vessel 202 may also include a processing solution (notshown) to effect a transformation on the target material when the targetmaterial enters the processing vessel 202. The processing solution (notshown) may be a preservative, a cell adhesion solution, a dye, or thelike. Unlike the displacement fluid 212, most, if not all, of theprocessing solution (not shown) remains within the processing vessel 202upon centrifugation, thereby effecting the transformation on the targetmaterial in one manner or another (i.e. preserving, increasing adhesionproperties, or the like). The processing solution (not shown) may beintroduced as a liquid or as a liquid contained in a casing. The casingmay be dissolvable in an aqueous solution but not in the displacementfluid 212 (such as gel cap); or, the casing may be breakable, such thatthe casing breaks when the processing vessel 202 is shaken in a vortexmixer. Additionally, more than one processing solution may be used.

The processing vessel 202 may include a flexible cap that can be pushedto dispense a pre-determined volume therefrom and onto a substrate, suchas a slide or a well plate. The cap 208 may be flexible or the cap 208may be removed and the flexible cap inserted into the open end 206.Alternatively, the processing vessel 202 may be attached to (i.e. afteraccumulating the target material) or may include a dispenser, which iscapable of dispensing a pre-determined volume of target material fromthe processing vessel 202 onto another substrate, such as a microscopeslide. The dispenser may repeatedly puncture the re-sealable cap 208 orcompress the material within the processing vessel 202 to withdraw anddispense the pre-determined volume of target material onto thesubstrate. Alternatively, the cap 208 may be removed and the dispenser(not shown) may be inserted directly into the processing vessel 202 todispense the buffy coat-processing solution mixture.

FIG. 3A shows an isometric view of a tube 300. FIG. 3B shows across-sectional view of the tube 300 taken alone the line III-III. Thetube 300 is similar to the tube 100 except that the tube 300 includes avalve 302 instead of a cannula 112 to provide a passageway between thefunnel 110 and the cavity 114. When undergoing centrifugation, the valve302 opens to permit fluid flow between the funnel 110 and the cavity114. When not undergoing centrifugation, the valve 302 is closed toinhibit fluid flow between the funnel 110 and the cavity 114. The valve302 may include but is not limited to a ball check valve, a diaphragmcheck valve, a swing check valve, a tilting disk check valve, a liftcheck valve, and a duckbill valve. Alternatively, the valve 302 isclosed when the centrifugal forces are less than or equal to apredetermined amount and the valve 320 is open when the centrifugalforces are greater than or equal to a predetermined amount. Thepredetermined amount may include, but is not limited to, 2 g, 5 g, 10 g,100 g, 1000 g, 2000 g, 2500 g, 3000 g, 5000 g, or 10000 g, where g isthe force of gravity.

The collection end 106 may include a break-point 304 where to permit thecavity 114 and the valve 302 to be separated from the funnel 110 so thatthe target material may be removed and retained in a single vessel forsubsequent processing. The break-point 304 may include threads, a tongueand groove joint, a dovetail joint, or any appropriate connectionmethod.

Before centrifugation and after inverting the tube 300, the plug 116 maybe removed from the cavity 114 and the cavity 114 may be loaded with thedisplacement fluid 212. The displacement fluid 212 displaces the targetmaterial, such that when the tube 300 undergoes centrifugation, thedisplacement fluid 212 flows out of the processing cavity 114 and intothe funnel 110 via the open valve 302, and, through displacement, suchas through buoyant displacement (i.e. lifting a material upwards),pushes the target material through the open valve 302 and into thecavity 114. During centrifugation, the cavity 114 may be sealed by acover (not shown) which may include threads, may be puncturable andresealable, or may be a one-time use, such as a foil.

FIG. 4A shows an isometric view of a tube 400. FIG. 4B shows across-sectional view of the tube 400 taken alone the line IV-IV. Thetube 400 is similar to the tube 100 except that the tube 400 includes ahole 402 instead of a cannula 112 to provide a passageway between thefunnel 110 and the cavity 114. The hole 402 has a diameter that is sizedto inhibit fluid communication between the funnel 110 and the cavity 114before and after centrifugation and to allow fluid communication betweenthe funnel 110 and the cavity 114 during centrifugation. The diameter,for example, may be based on the respective surface tensions of thedisplacement fluid 212 and the target material.

Before centrifugation and after inverting the tube 400, the plug 116 maybe removed from the cavity 114 and the cavity 114 may be loaded with thedisplacement fluid 212. The displacement fluid 212 displaces the targetmaterial, such that when the tube 400 undergoes centrifugation, thedisplacement fluid 212 flows out of the processing cavity 114 and intothe funnel 110 via the hole 402, and, through displacement, such asthrough buoyant displacement (i.e. lifting a material upwards), pushesthe target material through the 402 and into the cavity 114. Duringcentrifugation, the cavity 114 may be sealed by a cover (not shown)which may include threads, may be puncturable and resealable, or may bea one-time use, such as a foil.

Sealing Ring

FIG. 5A shows an isometric view of a sealing ring 500. FIG. 5B shows atop down view of the sealing ring 500. Dot-dashed line 502 representsthe central or highest-symmetry axis of the sealing ring 500. Thesealing ring 500 includes an inner wall 504, an outer wall 506, and acavity 508. In FIG. 5B, R_(IW) represents the radial distance from thecenter of the sealing ring 500 to the inner wall 504, and R_(OW)represents the radial distance from the center of the sealing ring 500to the outer wall 506. The sealing ring 500 is configured to fit arounda primary vessel, such as a tube. The cavity 508 is sized and shaped toreceive the primary vessel. The sealing ring 500 may be tightened, suchthat the size of the cavity 508 and the radii of the inner and outerwalls 504 and 506 are reduced by circumferentially applying anapproximately uniform, radial force, such as the radial force created bya clamp, around the outer wall 506 directed to the central axis 502 ofthe sealing ring 500. When the sealing ring 500 is tightened around theprimary vessel, the uniform force applied to the sealing ring 500 isapplied to the primary vessel, thereby causing the primary vessel toconstrict. When the radial force is removed from the sealing ring 500,the sealing ring 500 remains tightened and in tension around the primaryvessel.

The sealing ring may be any shape, including, but not limited to,circular, triangular, or polyhedral. FIG. 5C shows an isometric view ofa sealing ring 510. FIG. 5D shows a top down view of the sealing ring510. Sealing ring 510 is similar to sealing ring 500, except sealingring 510 is polyhedral. Dot-dashed line 512 represents the central orhighest-symmetry axis of the sealing ring 510. The sealing ring 510includes an inner wall 514, an outer wall 516, and a cavity 518. Thesealing ring may be composed of a metal, such as brass, a polymer, orcombinations thereof.

Alternatively, as shown in FIG. 5E, a sealing ring 520 may be composedof a piezoelectric material. FIG. 5F shows a top down view of thesealing ring 520. Dot-dashed line 522 represents the central orhighest-symmetry axis of the sealing ring 520. The sealing ring 520 maybe connected to an electric potential source 528, such as a battery, viaa first lead 524 and a second lead 526. The electric potential source528 creates a mechanical strain that causes the sealing ring 520 totighten (i.e. sealing ring 520 radii decrease). The sealing ring 520includes an inner wall 550, an outer wall 552, and a cavity 554. In FIG.5F, R_(IW) represents the radial distance from the center of the sealingring 520 to the inner wall 550, and R_(OW) represents the radialdistance from the center of the sealing ring 520 to the outer wall 552.Alternatively, the sealing ring 520 may be in a naturally tightenedstated. When applying the electric potential the sealing ring 520expands. Alternatively, a portion of the sealing ring may be composed ofthe piezoelectric material, such that the piezoelectric portion acts asan actuator to cause the other portion of the sealing ring to tightenand apply the substantially uniform circumferential pressure on theprimary vessel, thereby constricting the primary vessel to form theseal.

FIG. 5G shows an isometric view of a sealing ring 540. The sealing ringincludes an adjustment mechanism 548 to adjust the inner diameterR_(ID). The collapsible ring includes a first end 542 and a second end546, the first and second ends 542 and 546 being joined by a bandportion 544. The first and second ends 542 and 546 include complementaryportions of the adjustment mechanism 548. The adjustment mechanism 548includes, but is not limited to, a ratchet, tongue and groove, detents,or the like.

The sealing ring may also include a thermal element, such as a heatedwire. The thermal element may soften the primary vessel forconstriction. Alternatively, the thermal element may melt the primaryvessel to provide a more adherent seal. Alternatively, the thermalelement may cause the sealing ring to compress, thereby forming a sealbetween the primary vessel and float.

Methods

For the sake of convenience, the methods are described with reference toan example suspension of anticoagulated whole blood. But the methodsdescribed below are not intended to be so limited in their scope ofapplication. The methods, in practice, can be used with any kind ofsuspension. For example, a sample suspension can be urine, blood, bonemarrow, cystic fluid, ascites fluid, stool, semen, cerebrospinal fluid,nipple aspirate fluid, saliva, amniotic fluid, vaginal secretions, mucusmembrane secretions, aqueous humor, vitreous humor, vomit, and any otherphysiological fluid or semi-solid. It should also be understood that atarget material can be a fraction of a sample suspension, such as buffycoat, a cell, such as ova, fetal material (such as trophoblasts,nucleated red blood cells, fetal red blood cells, fetal white bloodcells, fetal DNA, fetal RNA, or the like), or a circulating tumor cell(“CTC”), a circulating endothelial cell, an immune cell (i.e. naïve ormemory B cells or naïve or memory T cells), a vesicle, a liposome, aprotein, a nucleic acid, a biological molecule, a naturally occurring orartificially prepared microscopic unit having an enclosed membrane,parasites (e.g. spirochetes, such as Borrelia burgdorferi which causeLyme disease; malaria-inducing agents), microorganisms, viruses, orinflammatory cells. Alternatively, the sample may be a biological solid,such as tissue, that has been broken down, such as by collagenase, priorto or after being added to the primary vessel.

For example, target material enrichment is a process by which the targetmaterials are purified relative to non-target material. For example, thetarget material may be enriched relative to non-target material, therebyhaving a ratio as low as 1 part target material, such as a single cell,protein, DNA, or the like, to 30,000,000 parts non-target material.Other ratios may include, but at not limited to, as low as approximately1:25,000,000, 1:15,000,000, 1:10,000,000, 1:5,000,000, 1:1,000,000,1:250,000, 1:100,000, 1:50,000, 1:25,000, 1:10,000, 1:1,000, 1:100,1:10, or 1:1.

Furthermore, for the sake of convenience, the method is described withreference to centrifugation, such as 2 g, 5 g, 10 g, 100 g, 1000 g, 1250g, 1500 g, 2000 g, 2500 g, 3000 g, 5000 g, or 10000 g, where g is theforce of gravity. But the methods described below are not intended to beso limited in their scope of application. For example, centrifugationmay not be used, and gravity (i.e. 1 g) may be used to permit theexchange of fluids and/or the separation of fluids. Alternatively, thedensities of the fluids described below may be so great or thedifferentiation between the densities of the fluids may be so great thatthe separation and/or exchange of fluids occur without centrifugation.The methods, even without centrifugation, may be performed in anyappropriate amount of time, including, but not limited to, less than onehour (i.e. 1 min, 5 min, 10 min, 15 min, 20 min, 30 min, 45 min, etc.),one hour (i.e. 60 min) or more than one hour (i.e. 90 min, 2 hours, 4hours, 8 hours, 24 hours, etc.).

Furthermore, in all methods, a filling device (not shown) may beinserted into the cavity 114 of the tube 100 prior to the processingvessel 202 being inserted into the cavity 114. The filling device (notshown), such as a pump or syringe, includes a layering fluid having adensity which may be less than or greater than the target material. Thecannula 112 extends into the filling device (not shown) to access aninner volume at least partially filled with the layering fluid. Thefilling device (not shown) is used to displace or remove or substituteair within the tube with the layering fluid, such as by undergoingcentrifugation or by providing a pressure gradient. The layering mayhave a density greater than or less than the target material. Examplesof suitable layering fluids include, but are not limited to, solution ofcolloidal silica particles coated with polyvinylpyrrolidone (e.g.Percoll), polysaccharide solution (e.g. Ficoll), iodixanol (e.g.OptiPrep), an organic solvent, a liquid wax, an oil, a gas, andcombinations thereof; olive oil, mineral oil, silicone oil, immersionoil, mineral oil, paraffin oil, silicon oil, fluorosilicone,perfluorodecalin, perfluoroperhydrophenanthrene, perfluorooctylbromide,and combinations thereof; organic solvents such as 1,4-Dioxane,acetonitrile, ethyl acetate, tert-butanol, cyclohexanone, methylenechloride, tert-Amyl alcohol, tert-Butyl methyl ether, butyl acetate,hexanol, nitrobenzene, toluene, octanol, octane, propylene carbonate,tetramethylene sulfones, and ionic liquids; polymer-based solutions;surfactants; perfluoroketones, such as perfluorocyclopentanone andperfluorocyclohexanone, fluorinated ketones, hydrofluoroethers,hydrofluorocarbons, perfluorocarbons, perfluoropolyethers, silicon andsilicon-based liquids, such as phenylmethyl siloxane; and combinationsthereof.

Method I

FIG. 6A shows a flow diagram for an example method for retrieving targetmaterial. In block 602, a suspension, such as anticoagulated wholeblood, is obtained. In block 604, the whole blood is added to a primaryvessel, such as a test tube. In block 606, a float is added to the tube.FIG. 7A shows a whole blood sample 702 and a float 704 added to the tube100. The float 704 includes a main body, two teardrop-shaped end caps,and support members radially spaced and axially oriented on the mainbody. Alternatively, the float 704 may not include any support members.Alternatively, the float 704 may include support members which do notengage the inner wall of the tube 100.

In alternative embodiments, the number of support members, supportmember spacing, and support member thickness can each be independentlyvaried. The support members can also be broken or segmented. The mainbody is sized to have an outer diameter that is less than the innerdiameter of the tube 100, thereby defining fluid retention channelsbetween the outer surface of the main body and the inner wall of thetube 100. The surfaces of the main body between the support members canbe flat, curved or have another suitable geometry. The support membersand the main body may be a singular structure or may be separatestructures.

Embodiments include other types of geometric shapes for float end caps.The top end cap may be teardrop-shaped, dome-shaped, cone-shaped, or anyother appropriate shape. The bottom end cap may be teardrop-shaped,dome-shaped, cone-shaped, or any other appropriate shape. In otherembodiments, the main body of the float 504 can include a variety ofdifferent support structures for separating samples, supporting the tubewall, or directing the suspension fluid around the float duringcentrifugation. Embodiments are not intended to be limited to theseexamples. The main body may include a number of protrusions that providesupport for the tube. In alternative embodiments, the number and patternof protrusions can be varied. The main body may include a singlecontinuous helical structure or shoulder that spirals around the mainbody creating a helical channel. In other embodiments, the helicalshoulder can be rounded or broken or segmented to allow fluid to flowbetween adjacent turns of the helical shoulder. In various embodiments,the helical shoulder spacing and rib thickness can be independentlyvaried. In another embodiment, the main body may include a supportmember extending radially from and circumferentially around the mainbody. In another embodiment, the support members may be tapered.

The float 704 can be composed of a variety of different materialsincluding, but not limited to, metals; organic or inorganic materials;ferrous plastics; sintered metal; machined metal; plastic materials andcombinations thereof. The tube 100 may have an inner wall and a firstdiameter. The float 704 can be captured within the tube 100 by aninterference fit, such that under centrifugation, an inner wall of thetube 100 expands to permit axial movement of the float 704. Whencentrifugation stops, the inner wall reduces back to the first diameterto induce the interference fit. Alternatively, the inner wall may notexpand and the interference fit may not occur between the float 704 andthe tube 100, such that the float moves freely within the tube before,during, or after centrifugation. The end caps of the float may bemanufactured as a portion of the main body, thereby being one singularstructure, by machining, injection molding, additive techniques, or thelike; or, the end caps may be connected to the main body by a press fit,an adhesive, a screw, any other appropriate method by which to hold atleast two pieces together, or combinations thereof.

Returning to FIG. 6A, in block 608, the primary vessel, the float, andthe whole blood undergo density-based separation, such as bycentrifugation, thereby permitting separation of the whole blood intodensity-based fractions along an axial position in the tube based ondensity. FIG. 7B shows an isometric view of the tube 100, the float 704,and the blood having undergone density-based separation, such as bycentrifugation. Suppose, for example, the centrifuged whole bloodincludes three fractions. For convenience sake, the three fractionsinclude plasma, buffy coat, and red blood cells. However, when anothersuspension undergoes centrifugation, there may be more than, less than,or the same number of fractions, each fraction having a differentdensity. The suspension undergoes axial separation into three fractionsalong the length the tube based on density, with red blood cells 714located on the bottom, plasma 710 located on top, and buffy coat 712located in between, as shown in FIG. 7B. The float 704 may have anyappropriate density to settle within one of the fractions. The densityof the float 704 can be selected so that the float 704 expands the buffycoat 712 between the main body of the float and the inner wall of theprimary vessel. The buffy coat 712 can be trapped within an area betweenthe float 704 and the primary vessel 702.

Returning to FIG. 6A, in block 610, the plasma 710 may be removed fromthe tube 100, such as by pipetting, suctioning, pouring, or the like. Inblock 612, as seen in FIG. 7C, at least one delineation fluid 720 may beadded to the tube. The delineation fluid may provide further separationbetween the target material and any non-target material above and/orbelow the target material. The at least one delineation fluid 720 mayhave a density greater than or less than the target material. Forexample, when it is desirous to further separate the buffy coat 712 andthe red blood cells 714, the delineation fluid may have a densitygreater than the buffy coat 712 and less than the red blood cells 714.The at least one delineation fluid 720 may be miscible or immisciblewith the suspension fluid and inert with respect to the suspensionmaterials. The at least one delineation fluid 720 may also provide anarea in which to seal the primary vessel 702, because there is greaterdelineation and separation between the buffy coat 712 and the red bloodcells 714. The at least one delineation fluid 720 may be used whether ornot a float is used. Examples of suitable delineation fluids include,but are not limited to, solution of colloidal silica particles coatedwith polyvinylpyrrolidone (e.g. Percoll), polysaccharide solution (e.g.Ficoll), iodixanol (e.g. OptiPrep), cesium chloride, sucrose,sugar-based solutions, polymer-based solutions, surfactants, an organicsolvent, a liquid wax, an oil, a gas, and combinations thereof; oliveoil, mineral oil, silicone oil, immersion oil, mineral oil, paraffinoil, silicon oil, fluorosilicone, perfluorodecalin,perfluoroperhydrophenanthrene, perfluorooctylbromide, and combinationsthereof; organic solvents such as 1,4-Dioxane, acetonitrile, ethylacetate, tert-butanol, cyclohexanone, methylene chloride, tert-Amylalcohol, tert-Butyl methyl ether, butyl acetate, hexanol, nitrobenzene,toluene, octanol, octane, propylene carbonate, tetramethylene sulfones,and ionic liquids; polymer-based solutions; surfactants;perfluoroketones, such as perfluorocyclopentanone andperfluorocyclohexanone, fluorinated ketones, hydrofluoroethers,hydrofluorocarbons, perfluorocarbons, perfluoropolyethers, silicon andsilicon-based liquids, such as phenylmethyl siloxane; and combinationsthereof.

In block 614, the tube, float, delineation fluid, and remainingfractions undergo re-centrifugation. In block 616, a sealing ring isapplied.

FIG. 7D shows a two-part seal including an internal pliant part to beinserted into the primary vessel and an external constricting part toconstrict and deform at least the primary vessel to seal an upperportion of the primary vessel from a lower portion of the primaryvessel. The external constricting part may also constrict and deform theinternal pliant part. The two-part seal prevents fluids from moving pastthe two-part seal within the primary vessel and prevents movement of theinternal pliant part. For the sake of convenience, the internal pliantpart is described with reference to the float 704 and the externalconstricting part is described with reference to the sealing ring 500,but the system described is not intended to be so limited and mayinclude any appropriate float and any appropriate external constrictingdevice.

The sealing ring 500 exerts circumferential or radial forces on the tube100, thereby causing the tube 100 to collapse inwardly against the float704. Magnified view 722 shows the sealing ring 500 tightened around thefloat 704 and the tube 100. The sealing ring 500, having been placed atan interface of the delineation fluid 720 and the red blood cells 714,causes the tube 100 to collapse inwardly until a seal is formed betweenthe tube 100 and the float 704. An outer wall of the sealing ring 500may sit flush with an outer wall of the tube 100; the outer wall of thesealing ring 500 may extend past the outer wall of the tube 100; or, theouter wall of the tube 100 may extend past the outer wall of the sealingring 500. The sealing ring 500 remains tightened to maintain the seal,which prevents fluids from moving past the seal in any direction. Thesealing ring 500 may also remain in tension. Alternatively, the sealingring 500 may be overtightened and then the force applied to the sealingring 500 is removed. The sealing ring 500 may expand slightly, thoughstill remains constricted.

To apply the sealing ring 500 and thereby form the seal, a clamp may beused to circumferentially apply a force directed toward the central axisof the tube 100 to the sealing ring 500. The sealing ring 500 is placedaround the tube 100 after the tube 100 undergoes density-basedseparation, such as by centrifugation. The sealing ring 500 and the tube100 are then placed into the clamp. The clamp may include a shelf tosupport the sealing ring 500 against the tube 100. Operation of theclamp may be automated or may be performed manually. Alternatively, theclamp may form a seal between the float 704 and tube 100 without theinclusion of the sealing ring 500. Alternatively, a seal may be formedbetween the float 704 and the tube 100 such as by ultrasonic welding; orby applying heat or a temperature gradient to deform and/or melt thetube 100 to the float 704. For the sake of convenience, the methods aredescribed with reference to the sealing ring, but the methods describedbelow are not intended to be so limited in their application and may beperformed without the sealing ring.

When operation of the clamp is automated, a motor causes translation ofeither a collet, including collet fingers, or a pressure member to causecompression of the collet fingers. The motor may be connected to thecollet or the pressure member by a shaft, such as a cam shaft, and oneor more gears. A base engages and holds the object. When the collet isdriven by the motor, the pressure member remains stationary. When thepressure member is driven by the motor, the collet remains stationary.The clamp may include a release, so as to cause the pressure member toslide off of the collet fingers 904, thereby removing the clampingforce.

Alternatively, the clamp may be, but is not limited to, a collet clamp,an O-ring, a pipe clamp, a hose clamp, a spring clamp, a strap clamp, ora tie, such as a zip tie. The clamp may be used without a sealing ringto provide a seal between a float and a tube.

Returning to FIG. 6A, in block 618, and as seen in FIG. 7E, the tube 100may be inverted, the plug 116 may be removed from the tube 100, theprocessing vessel 202 may be inserted into the tube 200, and thedisplacement fluid 212 may be added to the processing vessel 202. It benoted that the displacement fluid 212 may be added to the processingvessel 202 before or after inserting the processing vessel 202 into thetube 200

Returning to FIG. 6A, in block 620, the system is then re-centrifuged.FIG. 7F shows the tube 100 and the processing vessel 202 aftercentrifugation. As the displacement fluid 212, having a density greaterthan the buffy coat 712 but less than the delineation fluid 720, flowsfrom the processing vessel 202 into the tube 100, the buffy coat 712moves upwards within the tube 100 through the cannula 214, and into theprocessing vessel 202.

The processing vessel 202 including the buffy coat 712 may then beremoved from the tube 100 to undergo further processing, analysis,storage, or the like. After removing the processing vessel 202, aprocessing solution may be added, though the processing solution mayhave already been in the processing vessel prior to retrieval of thetarget material. The processing vessel may be shaken, such as by avortex mixer. The processing solution (not shown), having been addedbefore shaking either in liquid form, in a dissolvable casing, or in abreakable casing, may then mix with the buffy coat to effect atransformation and form a buffy coat-processing solution mixture. Thebuffy coat-processing solution mixture may then be dispensed onto asubstrate, such as a microscope slide.

Method II

FIG. 6B shows a flow diagram for an example method for retrieving targetmaterial. In block 632, a suspension, such as anticoagulated whole blood702, is obtained. In block 634, and as seen in FIG. 8A, the whole blood702 is added to the tube 100. Returning to FIG. 6B, in block 636, and asseen in FIG. 8B, a stain 806, a negative enrichment reagent 804, and adelineation fluid 802 may be added to the tube 100. The stain 806 isadded to the primary vessel to label the target material. The negativeenrichment reagent 804 is also added to the primary vessel to change thedensity of a non-target material, for example, the plasma 510, withoutchanging the density of the target material. For example, the negativeenrichment reagent 804 may change the density of a non-target materialsuch that the density of the target material is less than or greaterthan a changed density of the non-target material. The stain 806 and thenegative enrichment reagent 804 may be allowed to incubate for anyappropriate amount of time, including, but not limited to, less than onehour (i.e. 1 min, 5 min, 10 min, 15 min, 20 min, 30 min, 45 min, etc.),one hour (i.e. 60 min) or more than one hour (i.e. 90 min, 2 hours, 4hours, 8 hours, 24 hours, etc.).

During incubation, the tube 100 may be vortexed, rocked, shaken, or anyappropriate movement to enhance mixing of the stain with the targetmaterial. A stain, for example, may be a solution containing afluorescent probe may be used to stain and thereby label the targetmaterial, thereby providing a fluorescent signal for identification andcharacterization. The solution containing the fluorescent probe may beadded to the suspension before the suspension is added to the vessel,after the suspension is added to the vessel but before centrifugation,or after the suspension has undergone centrifugation. The fluorescentprobe includes a fluorescent molecule bound to a ligand. The targetmaterial may have a number of different types of surface markers. Eachtype of surface marker is a molecule, such an antigen, capable ofattaching a particular ligand, such as an antibody. As a result, ligandscan be used to classify the target material and determine the specifictype of target materials present in the suspension by conjugatingligands that attach to particular surface markers with a particularfluorescent molecule. Examples of suitable fluorescent moleculesinclude, but are not limited to, quantum dots; commercially availabledyes, such as fluorescein, FITC (“fluorescein isothiocyanate”),R-phycoerythrin (“PE”), Texas Red, allophycocyanin, Cy5, Cy7, cascadeblue, Hoechst, DAPI (“4′,6-diamidino-2-phenylindole”) and TRITC(“tetramethylrhodamine isothiocyanate”); combinations of dyes, such asCY5PE, CY7APC, and CY7PE; and synthesized molecules, such asself-assembling nucleic acid structures. Many solutions may be used,such that each solution includes a different type of fluorescentmolecule bound to a different ligand. Furthermore, a nucleus of thetarget material may have a different size than the nucleuses of thenon-target material. Determining nuclear size may aid in differentiatingbetween target and non-target material.

Returning to FIG. 6B, in block 638, and as seen in FIG. 8D, the tube 100may be inverted, the plug 116 may be removed from the tube 100, theprocessing vessel 202 may be inserted into the tube 200, and thedisplacement fluid 212 may be added to the processing vessel 202. It benoted that the displacement fluid 212 may be added to the processingvessel 202 before or after inserting the processing vessel 202 into thetube 200

Returning to FIG. 6B, in block 640, the system is then re-centrifuged.FIG. 8E shows the tube 100 and the processing vessel 202 aftercentrifugation. As the displacement fluid 212, having a density greaterthan the buffy coat 712 but less than the delineation fluid 720, flowsfrom the processing vessel 202 into the tube 100, the buffy coat 712moves upwards within the tube 100 through the cannula 214, and into theprocessing vessel 202. Furthermore, the density-alerting fluid 804,having mixed with the plasma 710 to form a high density plasma 808,causes the high density plasma 808 to have a density greater than atleast the buffy coat 702 and the delineation fluid 802.

The processing vessel 202 including the buffy coat 712 may then beremoved from the tube 100 to undergo further processing, analysis,storage, or the like. After removing the processing vessel 202, aprocessing solution may be added, though the processing solution mayhave already been in the processing vessel prior to retrieval of thetarget material. The processing vessel may be shaken, such as by avortex mixer. The processing solution (not shown), having been addedbefore shaking either in liquid form, in a dissolvable casing, or in abreakable casing, may then mix with the buffy coat to effect atransformation and form a buffy coat-processing solution mixture. Thebuffy coat-processing solution mixture may then be dispensed onto asubstrate, such as a microscope slide.

Examples of suitable negative enrichment reagents include, but are notlimited to, a solution of colloidal silica particles coated withpolyvinylpyrrolidone (e.g. Percoll), polysaccharide solution (e.g.Ficoll), iodixanol (e.g. OptiPrep), a complex, branch glucan (e.g.Dextran), cesium chloride, sucrose, sugar-based solutions, polymersolutions, multi-phase polymer solutions, tetrameric antibody complexes(e.g. RosetteSep) or the like; or particles, such as beads (composed ofat least one of a metal, silica, glass, a polymer, or the like),nanoparticles, metal-based compounds, metal complexes, lipids, sugars,or the like.

The positive enrichment reagent is also added to the primary vessel tochange the density of the target material without changing the densityof a non-target material. For example, the positive enrichment reagentmay change the density of the target material such that the density ofthe non-target material is less than or greater than a changed densityof the target material. The positive enrichment reagent may be allowedto incubate for any appropriate amount of time, including, but notlimited to, less than one hour (i.e. 1 min, 5 min, 10 min, 15 min, 20min, 30 min, 45 min, etc.), one hour (i.e. 60 min) or more than one hour(i.e. 90 min, 2 hours, 4 hours, 8 hours, 24 hours, etc.). Examples ofsuitable positive enrichment reagents include, but are not limited to, asolution of colloidal silica particles coated with polyvinylpyrrolidone(e.g. Percoll), polysaccharide solution (e.g. Ficoll), iodixanol (e.g.OptiPrep), a complex, branch glucan (e.g. Dextran), cesium chloride,sucrose, sugar-based solutions, polymer solutions, multi-phase polymersolutions, tetrameric antibody complexes (e.g. RosetteSep) or the like;or particles, such as beads (composed of at least one of a metal,silica, glass, a polymer, or the like), nanoparticles, metal-basedcompounds, metal complexes, lipids, sugars, or the like.

Fraction Retrieval

In one instance, it may be desirable to remove a full fraction of theblood sample to obtain the target material.

In another instance, it may be desirable to remove a portion of afraction of the blood sample to obtain the target material. Sequentialdensity fractionation is the division of a sample into fractions or of afraction of a sample into sub-fractions by a step-wise or sequentialprocess, such that each step or sequence results in the collection orseparation of a different fraction or sub-fraction from the precedingand successive steps or sequences. In other words, sequential densityfractionation provides individual sub-populations of a population orindividual sub-sub-populations of a sub-population of a populationthrough a series of steps. For example, buffy coat is a fraction of awhole blood sample. The buffy coat fraction can be further broken downinto sub-fractions including, but not limited to, reticulocytes,granulocytes, lymphocytes/monocytes, and platelets. The buffy coat mayalso contain the desired target material. These sub-fractions may beobtained individually by performing sequential density fractionation.

Two or more processing vessels and respective displacement fluids may beused depending on the number of fractions or sub-fractions desired forseparation and collection. Each successive displacement fluid is denserthan the preceding displacement fluid. Furthermore, the displacementfluid to collect the target material has a density greater than thedensity of the desired target material; for example, example, thedisplacement fluid may have a density that is approximately 0.001 toapproximately 0.005 g/cm³ greater than the density of the desired targetmaterial. Similarly, each successive fraction or sub-fraction is denserthan the preceding fraction or sub-fraction. Once collected, theconsecutive sub-fractions may be analyzed, such as for diagnostic,prognostic, research purposes, to determine components characteristics(i.e. a complete blood count), how those characteristics change overtime, or the like.

Subsequent processing vessels and displacement fluids may be used tocollect additional subfractions of the buffy coat 712 until allsubfractions are collected or until the desired subfraction iscollected. Though sequential density fractionation is described as beingperformed with a float and a sealing ring, sequential densityfractionation may be performed without a float, a sealing ring, or both.The following is an example method for performing sequential densityfractionation after having performed all steps up to insertion of theprocessing vessel:

-   -   1. inserting an (n−y)^(th) processing vessel into a cavity        within the tube,    -   2. adding an (n−y)^(th) displacement fluid to the (n−y)^(th)        processing vessel, the (n−y)^(th) displacement fluid;    -   3. centrifuging the primary vessel and the (n−y)^(th) processing        vessel together, the (n−y)^(th) displacement fluid to flow into        the primary vessel via the tube to displace an (n−y)^(th)        sub-fraction of the suspension from the primary vessel into the        (n−y)^(th) processing vessel through the tube via the cannula;    -   4. removing the (n−y)^(th) processing vessel including the        (n−y)^(th) sub-fraction from the tube.        For example, n may be any number greater than or equal to 1 and        y may be any number greater than or equal to 0, such as n=2 and        y=1. Furthermore, n may be the total number of sub-fractions        desired and y=(n−1-number of sub-fractions already collected).

After retrieving the target material, the target material may be placedon a substrate for imaging and detection (and subsequent storage forarchival purposes). At least one portion of the detected target materialmay then be removed from the substrate, such as by picking, to undergofurther analysis. The isolated target material may be deposited into aPCR tube, a well of a well plate, a slide, or any appropriate substrateor vessel for performing the further analysis.

The target material may be analyzed using any appropriate analysismethod or technique, though more specifically extracellular andintracellular analysis including intracellular protein labeling;chromogenic staining; nucleic acid analysis, including, but not limitedto, DNA arrays, expression arrays, protein arrays, and DNA hybridizationarrays; in situ hybridization (“ISH”—a tool for analyzing DNA and/orRNA, such as gene copy number changes); polymerase chain reaction(“PCR”); reverse transcription PCR; or branched DNA (“bDNA”—a tool foranalyzing DNA and/or RNA, such as mRNA expression levels) analysis. Theisolated nucleated red blood cell may undergo further processing to testfor such fetal abnormalities including, but not limited to, chromosomalabnormalities (e.g. fetal aneuploidy, Down syndrome, trisomy 13, trisomy18, or a sex chromosome abnormality, such as Turner syndrome), smallersub-chromosomal abnormalities, gender testing, mutational analysis, andrhesus blood type testing.

These techniques may require fixation, permeabilization, and isolationof the target material prior to analysis. Some of the intracellularproteins which may be labeled include, but are not limited to,cytokeratin (“CK”), actin, Arp2/3, coronin, dystrophin, FtsZ, myosin,spectrin, tubulin, collagen, cathepsin D, ALDH, PBGD, Akt1, Akt2, c-myc,caspases, survivin, p27^(kip), FOXC2, BRAF, Phospho-Akt1 and 2,Phospho-Erk1/2, Erk1/2, P38 MAPK, Vimentin, ER, PgR, PI3K, pFAK, KRAS,ALKH1, Twist1, Snail1, ZEB1, Fibronectin, Slug, Ki-67, M30, MAGEA3,phosphorylated receptor kinases, modified histones, chromatin-associatedproteins, and MAGE. To fix, permeabilize, or label, fixing agents (suchas formaldehyde, formalin, methanol, acetone, paraformaldehyde, orglutaraldehyde), detergents (such as saponin, polyoxyethylene,digitonin, octyl β-glucoside, octyl β-thioglucoside,1-S-octyl-β-D-thioglucopyranoside, polysorbate-20, CHAPS, CHAPSO,(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol or octylphenolethylene oxide), or labeling agents (such as fluorescently-labeledantibodies, enzyme-conjugated antibodies, Pap stain, Giemsa stain, orhematoxylin and eosin stain) may be used.

The density of the target material may be increased (such as byattaching a weight to the target material or by having the targetmaterial absorb or ingest the weight) or may be decreased (such as byattaching a buoy to the target material or by having the target materialabsorb or ingest the buoy). The weight or the buoy may be bound to aligand. The target material may have a number of different types ofsurface markers. Each type of surface marker is a molecule, such as anantigen, capable of attaching a particular ligand, such as an antibody.As a result, ligands can be selected to attached specifically to thetarget material to alter the density of the target material. Examples ofsuitable weights and/or buoys include, but are not limited to beadscomposed of metal, glass, ceramic, plastic, or combinations thereof.Alternatively, the weight or buoy may be attached to a non-targetmaterial to change the density of the non-target material to obtain apurer sample of the target material.

The foregoing description, for purposes of explanation, used specificnomenclature to provide a thorough understanding of the disclosure.However, it will be apparent to one skilled in the art that the specificdetails are not required in order to practice the systems and methodsdescribed herein. The foregoing descriptions of specific embodiments arepresented by way of examples for purposes of illustration anddescription. They are not intended to be exhaustive of or to limit thisdisclosure to the precise forms described. Many modifications andvariations are possible in view of the above teachings. The embodimentsare shown and described in order to best explain the principles of thisdisclosure and practical applications, to thereby enable others skilledin the art to best utilize this disclosure and various embodiments withvarious modifications as are suited to the particular use contemplated.It is intended that the scope of this disclosure be defined by thefollowing claims and their equivalents:

I/We claim:
 1. A system comprising: a tube comprising a first end, asecond end comprising an aperture, a biological suspension comprising atarget material, and a collection segment between the first and secondends, the collection segment comprising a funnel comprising a mouthproximal to the first end and an apex distal to the first end, a cavityextending from the aperture in the second end towards the apex of thefunnel, and a cannula extending from the apex of the funnel into thecavity; and a processing vessel comprising a closed end and adisplacement fluid having a density greater than a density of the targetmaterial, wherein at least a portion of the processing vessel is locatedwithin the cavity of the tube, and wherein the cannula extends throughthe closed end of the processing vessel.
 2. The system of claim 1,wherein the displacement fluid is selected from the group consisting of:a solution of colloidal silica particles coated withpolyvinylpyrrolidone, a polysaccharide solution, iodixanol, an organicsolvent, a liquid wax, an oil, a gas, olive oil, mineral oil, siliconeoil, immersion oil, mineral oil, paraffin oil, silicon oil,fluorosilicone, perfluorodecalin, perfluoroperhydrophenanthrene,perfluorooctylbromide, organic solvents, 1,4-Dioxane, acetonitrile,ethyl acetate, tert-butanol, cyclohexanone, methylene chloride,tert-Amyl alcohol, tert-Butyl methyl ether, butyl acetate, hexanol,nitrobenzene, toluene, octanol, octane, propylene carbonate,tetramethylene sulfones, ionic liquids, a polymer-based solution, asurfactant, a perfluoroketone, perfluorocyclopentanone,perfluorocyclohexanone, a fluorinated ketone, a hydrofluoroether, ahydrofluorocarbon, a perfluorocarbon, a perfluoropolyether, silicon, asilicon-based liquid, phenylmethyl siloxane, and combinations thereof.3. The system of claim 1, the processing vessel further comprising aresealable plug in the closed end, wherein the cannula extends throughthe resealable plug.
 4. The system of claim 1, wherein the cannula is atube, a needle, or a non-coring needle.
 5. The system of claim 1, theprocessing vessel further comprising a processing solution to effect atransformation on the target material.
 6. The system of claim 1, thebiological suspension further comprising a non-target material and thetube further comprising a negative enrichment reagent to change thedensity of the non-target material.
 7. The system of claim 6, whereinthe suitable negative enrichment reagent is a solution of colloidalsilica particles coated with polyvinylpyrrolidone, a polysaccharidesolution, iodixanol, a branch glucan, cesium chloride, sucrose, asugar-based solution, a polymer solution, a multi-phase polymersolution, a tetrameric antibody complex, a particle, a bead, ananoparticle, a metal-based compound, a metal complex, a lipid, or asugar.
 8. The system of claim 1, further comprising a cap insertedwithin the first end of the tube.
 9. The system of claim 8, furthercomprising a clip placed over the cap and the first end of the tube. 10.The system of claim 1, further comprising a float located at alongitudinal position within the tube.
 11. The system of claim 10,further comprising a sealing ring located circumferentially around thetube at the same longitudinal position as at least a portion of thefloat within the tube.
 12. The system of claim 1, wherein the first endof the tube is a bottom end, the second end of the tube is a top end,and the processing vessel extends upwardly from the second end of thetube.
 13. The system of claim 1, wherein the target material is buffycoat, a cell, an ova, fetal material, a trophoblast, a nucleated redblood cell, a fetal red blood cell, a fetal white blood cell, fetal DNA,fetal RNA, a circulating tumor cell, a circulating endothelial cell, animmune cell, a vesicle, a liposome, a protein, a nucleic acid, abiological molecule, a naturally occurring microscopic unit having anenclosed membrane, an artificially prepared microscopic unit having anenclosed membrane, a parasite, a microorganism, a virus, an inflammatorycell, or a broken down biological solid.
 14. The system of claim 1, thetube further comprising a positive enrichment reagent to change thedensity of the target material.
 15. The system of claim 14, wherein thesuitable positive enrichment reagent is a solution of colloidal silicaparticles coated with polyvinylpyrrolidone, a polysaccharide solution,iodixanol, a branch glucan, cesium chloride, sucrose, a sugar-basedsolution, a polymer solution, a multi-phase polymer solution, atetrameric antibody complex, a particle, a bead, a nanoparticle, ametal-based compound, a metal complex, a lipid, or a sugar.
 16. A methodcomprising the steps of: providing a tube comprising a first end, asecond end comprising an aperture, a biological suspension comprising atarget material, and a collection segment between the first and secondends, the collection segment comprising a funnel comprising a mouthproximal to the first end and an apex distal to the first end, a cavityextending from the aperture in the second end towards the apex of thefunnel, and a cannula extending from the apex of the funnel into thecavity; inserting a processing vessel comprising a closed end into thecavity of the tube, wherein the cannula extends through the closed endof the processing vessel; adding a displacement fluid having a densitygreater than a density of the target material to the processing vessel;and centrifuging the tube, the displacement fluid, and the processingvessel, wherein during centrifuging, the displacement fluid flows intothe tube via the cannula to displace the target material from the tubewhich flows into the processing vessel through the cannula.
 17. Themethod of claim 16, further comprising: adding a positive enrichmentreagent to the tube to change the density of the target material. 18.The method of claim 16, wherein the target material is buffy coat, acell, an ova, fetal material, a trophoblast, a nucleated red blood cell,a fetal red blood cell, a fetal white blood cell, fetal DNA, fetal RNA,a circulating tumor cell, a circulating endothelial cell, an immunecell, a vesicle, a liposome, a protein, a nucleic acid, a biologicalmolecule, a naturally occurring microscopic unit having an enclosedmembrane, an artificially prepared microscopic unit having an enclosedmembrane, a parasite, a microorganism, a virus, an inflammatory cell, ora broken down biological solid.
 19. The method of claim 16, furthercomprising: adding a negative enrichment reagent to the tube to changethe density of a non-target material the biological suspension furthercomprising a non-target material, the biological suspension furthercomprising the non-target material.
 20. The method of claim 16, furthercomprising the steps of: inserting a filling device comprising a closedend and a layering fluid into the cavity of the tube, wherein thecannula extends through the closed end of the filling device;transferring at least a portion of the layering fluid from the fillingdevice to the tube to displace air from the tube into the fillingdevice; and removing the filling device from the cavity of the tube,wherein the inserting, transferring, and removing steps of the fillingdevice are performed before inserting the processing vessel into thecavity of the tube.